Plant Biosystems, 134 (l) 61 -66.2000

On the taxonomy and distribution of Paeonia mascula s.l. in Italy based on rDNA ITS1 sequences

A. MUSACCHIO, G. PELLEGRINO. L. BERNARDO, N.G. PASSALACQUA and G. CESCA

received 15 March 1999; revised version accepted 23 July 1999

ABSTRACT - The internal transcribed spacers (ITS1) of nuclear ribosomal DNA of ten populations of Italian subspecies of Paeonia mascula s.l. were analysed. The aim was to correlate molecular and morphological data in order to solve taxonomic and distributive problems. Three highly homogeneous patterns of molecular variability and three corresponding groups of populations were identified. One group includes plants with both molecular and morphological similarities suggesting their attribution to P. mascula ssp. russoi. The other two groups show differences which do not grant their present separation in the two subspecies P. mascula ssp. mascula and P. mascula ssp. hellenica. This study confirms that reticulate evolution obscured morphological variability in Paeonia and stresses the usefulness of ITS analysis in studies at low taxonornic circumscriptions.

KEY WORDS - Paeonia mascula s.l., rDNA, ITS1 sequences, taxonomy

Paeonia mascula (L.) Miller sensu lato (Fam. Paeoniaceae, sect. Paeon, subsect. Foliolatae) is a perennial rhizomatous herb, with large dissected leaves and tomentose carpels. This species of European distribution is divided into four subspecies; P. mascula (L.) Miller ssp. mascula, found from central France to eastern Greece; P. mascula ssp. russoi (Biv.) Cullen et Heyw., from central Spain to western Greece; P. mascula ssp. hellenica Tzanoudakis, reported for southern Greece; P. mascula ssp, triternata (Pallas ex DC.) Stearn & Davis, reported for Rumania and the Crimean peninsula (Akeroyd, 1993; Greuter et al., 1984).

In Italy, reports indicate the presence of P. mascula ssp. mascula in the central and southern Apennines and Sicily, and of P. mascula ssp. russoi in Sicily, Sardinia and Calabria (Pignatti, 1982). Moreover, for Sicily, P. flavescens Presl (Presl, 1826), subsequently named P. corallina var. flavescens Guss. (Gussone, 1844), was also described. Stearn & Davis (1984) consider this taxon synonymous with P. mascula ssp. hellenica, implying in this manner the presence in Sicily of this latter species. The synonymy of P. mascula ssp. hellenica with P. flavescens is accepted also by Flora Europaea (Akeroyd, 1993) and Med Checklist (Greuter et al, 1984) but, surprisingly, the presence of P. mascula ssp. hellenica in Sicily is not reported in either of them. Recent phylogenetic studies on Paeonia, using the sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA, have provided evidence in favour of reticulate evolution in the section Paeon (Sang el al., 1995, 1997). The hybrid species were identified by full or partial nucleotide additivity detected at the sites that are variable in the sequences of the putative parental species. Species of hybrid origin predominate in the Mediterranean region; one of them is P. mascula s.l. with its subspecies (SANG el al., 1995). Interestingly, evidence provided by Sang et al. (1995) supports the contention that P. mascula ssp. mascula and P. mascuta ssp. hellenica share the same parental species, i.e., P. lactiflora and P. japonica or P obovata, while P. mascula ssp. russoi would derive from P lactiflora and P. mairei. Furthermore, the ITS1 sequences found in the three Italian subspecies show differences which could represent a tool to discriminate them. ITS variability, in fact, is commonly used to resolve relationships among close species or populations of the same species (Baldwin et al., 1995; Bellarosa et al., 1997).

For these reasons, and in the light of the uncertainty regarding the taxonomic status and distributional patterns of the Italian subspecies of P mascula s.l., specimens from various Italian populations were investigated for ITS1 sequence variability. The main purposes of this research were:

a) to discriminate among Italian accessions according to their ITS1 sequence variability and to compare the resulting patterns with those reported by SANG et al. (1995);

b) to establish a correlation between molecular data and morphological features, and desume taxonomical implications.

Materials and Methods

FIGURE 1 - Distribution of the 10 populations examined. Abbreviations of populations are as follows: Monte Albo (AL), Monte d'Iscudo (IS), Bruncu Spina (BS), Massiccio del Pollino (PO), Gargano (GA), Colli Albani (CA) Monti Iblei (IB), Monti Nebrodi (NE), Etna (ET) and Monti delle Madonie (MA).

One plant for each of the ten populations distributed in central and southern areas of the Italian territory, both peninsular and insular (Figure 1; Table 1), was examined, and all plants are presently cultivated in the Botanical Garden of the University of Calabria. Fresh cauline leaves (approx. 0.5 g) of individual plants were frozen in liquid nitrogen and ground into a fine powder. Total DNAs were then extracted according to CAPUTO et al. (1991). The ITS1 region was amplified by polymerase chain reaction (PCR) using two primers (5'-ATC C'l'G CAA TTC ACA CCA AGT ATC G-3'; 5'-GGA GAA GTC CTA ACA AGG TTT CCG-3'). All PCR reactions were conducted in a thermal cycler (30 cycles; 94°C 30", 55°C 60", 72°C 45"); the samples were denatured 3 min before the first cycle, and extension was prolonged for 7 min at the end of the last cycle. Sequencing reactions were carried out according to the enzymatic protocol using 10 ng DNA, resuspended in 6 nl tormamide and electrophoresed on a high resolution gel (4.75% polyacrylamide and 8 M urea).

Results

The ITS1 of all the specimens sequenced was 267 bp long, with a G+C content ranging from 56.1 to 57.6%. When sequences were compared, three different patterns were detected and the samples were arranged according to them in three groups characterised by a high degree of identity (Table 2). More precisely, a group (100% identity) includes all the samples from Sardinia (BS, AL, IS) along with that from M. Iblei; a second group (100% identity) includes specimens from the populations of Pollino (PO), Etna (ET) and Madonie (MA); the third group includes samples from Nebrodi (NE), Colli Albani (CA) and Gargano (GA) (99,6% identity). The degree of identity between the latter two groups was 99%, while Sardinian peonies exhibit 98% identity with the other groups. The ITS1 sequences obtained here have equal lengths, similar G+C contents and a smaller number of additive sites when compared to the ITS1 sequences of Paeonia mascula s.l. investigated by SANG et al. (1995). When the sequences of plants from the above Italian localities were compared with those oi the parental species, it was observed that the group from Sardinia and M. Iblei exhibits the same sequence as P. mairei (Table 3), while the other t\vo groups differ only in a few positions from the sequence of P. japonica. In particular, the group from Pollino has two bases which differ from those at the same positions in both its parental species (Table 4), while the group from M. Nebrodi shows a few additive sites, each combining with the correspondent nucleoticie of P. japonica (Table 4).

However, alignments of our sequences with those obtained by Sang el at. (1995) indicate that accessions from Sardinia and Iblei correspond to the molecular variability of P. russoi accessions from Pollino, Madonie and Etna fall into the molecular variability of P. mascula ssp. hellenica, and accessions from Nebrodi, Colli Albani and Gargano into the variability of P. mascula ssp. mascula.

DISCUSSION

The usefulness as well as the correct interpretation of the data obtained by sequencing ITS1, or any other multigene family, depends upon the level of homogeneity of the DNA repeat sequences produced by concerted evolution (Avise, 1994). When homogenisation does not occur, the broad variations of the copies within individuals may strongly reduce the potential in terms of phylogenetic reconstruction (Hillis et al., 1996).

The populations of P. mascula s.l. examined here show a more elevated level of homogenisation than previously examined populations (SANG et al., 1995), and thus may have undergone a higher rate of concerted evolution. This may have occurred as a consequence of special mating systems or of different ploidy levels among populations (Arnheim, 1983; Baldwin et al., 1995). Within Paeonia, the highest intragenomic uniformity for ITS1 sequences has been detected for the diploid P. cambessedesii (SANG et al.,

1997). In this regard, it is interesting to note that accessions from Sardinia and M. Iblei belong to diploid (2n=10) populations (Cesca et al., unpub.), differently from the sample analysed by Sang et al. (1995) which is tetraploid (2n=20) and of uncertain origin.

One of the aims of this research was to evaluate the taxonomic implications of molecular data and, in this perspective, it was necessary to evaluate the congruence between the molecular information and the morphological features of the individuals included in each of the groups identified on a molecular basis. The

group from Sardinia and M. Iblei, which is clearly distinguishable on a molecular basis and falls in the ITS1 variability of P. mascula ssp. russoi (SANG et al, 1995), includes plants with the same constant morphological features. Furthermore, these characters,

e.g. coriaceous and markedly reddish stem, leaves with long coiled hairs on the abaxial surface, allow a clear discrimination of the group from all the other plants examined. These populations belong to a separate taxon, which, also according to the information provided in both Flora Europaea and Med Checklist,

should be attributed to P. mascula ssp. russoi.

The remaining accessions can be arranged into two groups, each with an elevated molecular homology which, however, do not correspond in terms of morphological features. In fact, the group from Pollino, Madonie and Etna falls into the molecular variability of P. mascula ssp. bellenica (SANG et al., 1995), but includes individuals with flowers which are either white (typical of ssp. hellenica), or purple red (typical of ssp. mascula). Similarly, the group including specimens from Nebrodi, Colli Albani and Gargano falls within the molecular variability of P. mascula ssp. mascula, but the sample from Nebrodi produces flowers which are pink in colour.

These difficulties confirm the fact that molecular and morphological data are not always congruent in Paeonia (SANG ct al., 1997). Also, the distinction between ssp. mascula and ssp. hellenica, based only on flower colour, seems to be artificial. Probably, the

white flowered peonies, frequently found on the Sicilian mountains, represent a variability occurring in P. mascula ssp. mascula. This agrees with the observation that in Sicily white, red or light pink

flowers exist within single populations of Paeonia mascula s.l.

In conclusion, the molecular data gathered in this study suggest that the populations of P. mascula s.l. examined can be attributed to two clearly distinct taxa. One of them can be referred to P. mascula ssp. russoi (future studies will, however, clarify the correct nomenclature and taxonomic rank), which is widely diffused in Sardinia, while in Sicily it has been found in a site different from the locus classicus. The second group encompasses several Italian populations, originating from the same parental species, but with molecular and morphological differences which appear inadequate to separate the subspecies P. mascula ssp. mascula and P. mascula ssp. hellenica. As a consequence, in order to assess the presence in Sicily of P. mascula ssp. hellenica, further studies are needed, also on provenances from the locus classicus of this subspecies.

In conclusion, this study allowed us to emphasise minor differences within P. mascula s.l. and, at the same time, reinforced the idea that reticulate evolution has obscured the morphological variability in Paeonia.

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AUTHORS

Aldo Musacchiu (corresponding author), Giuseppe Pellegrino, Dipartimento di Ecologia, Universitá della Calabria, via P. Bucci, 87030 Arcavacata di Rende (CS), Italy

Liliana Bernardo, Nicodemo Giuseppe Passalacqua, Giuliano Cesca, Orto Botanico, Università della Calabria, 87030 Arcavacata di Rende (CS), Italy